Protein and microRNA (miRNA) repressers are present throughout eukaryotes, including humans, and regulate a variety of developmental processes. We want to understand how protein repressers and miRNAs work together to regulate an mRNA target. Specifically, we found the regulatory protein, Staufen, may function directly with a family of miRNAs, the mir-64 family, to regulate the fem-3 mRNA in C. elegans. Our goal is to address the following specific aims: 1. What roles does Staufen play in C. elegans development? 2. Does Staufen affect mRNA regulation via a miRNA? We will employ a combination of genetics, molecular biology, and biochemistry in the model organism C. elegans to further investigate the role of Staufen and miRNAs in mRNA regulation. To study the effects of Staufen and the mir-64 family on fem-3 regulation and development, we will analyze deletion mutants for each. We will utilize reporter transgenes to determine if Staufen and the mir-64 family of miRNAs affect fem-3 regulation. We will also determine when and where Staufen is expressed by doing Northerns, Westerns, and protein localization assays to characterize Staufen and make possible links to other known proteins or pathways. Immunoprecipitation (IP) experiments will be performed to confirm that Staufen interacts with the fem-3 mRNA and to verify that a miRNA is required for this interaction. Completion of the above project will aid our overall understanding of protein and miRNA repressers and will impact how we think about the regulation of developmental processes. Both protein and miRNA repressers are involved in developmental processes including embryogenesis, cardiogenesis, and myogenesis and have been linked to disease states such as cancer. The connection between protein and miRNA regulation will bring new insight into how these classes of molecules can coordinate developmental regulation and how defects in this regulation can lead to disease.